Neuroblastoma (NB) remains one of the most difficult pediatric cancers to treat, as nearly two-thirds of the patients present with metastasis at the tim of diagnosis. Despite aggressive treatment protocols, `high-risk' NB, stage 4 tumors in children older than 18 months of age and/or MYCN amplification, have dismal overall survival of only 50%. There is a significant gap in the comprehensive understanding of the tumor biology for tumor refractoriness and disease relapse, and therefore, elucidation of signaling mechanisms responsible for the aggressive tumor behavior would be highly significant for development of novel targeted clinical therapies. Members of the G-protein coupled receptor (GPCR) superfamily represent the hub of drug development activities and accounts for 40% of all targeted therapies. Our laboratory has previously shown that targeting one such GPCR, gastrin-releasing peptide receptor (GRP-R) and its ligand GRP inhibited NB tumorigenicity via regulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, a key cell survival pathway. During the previous funding period (see Progress Report), we reported that the GRP-R signaling regulates NB metastasis, specifically through activation of AKT2, and downregulation of PTEN, an endogenous negative regulator of AKT. The exact role of the GRP-R signaling and specific AKT isoform, AKT2, in the multi-step NB progression from tissue invasion to distant organ metastasis is yet to be elucidated. Targeting PI3K/AKT2, as well as mTOR, downstream of AKT, axis may be a potential strategy as this pathway regulates tumor vasculature and microenvironment (TME). In this competitive renewal application, we plan to determine the exact mechanisms by which GRP-R/AKT2 axis regulates the formation and maintenance of metastasis-initiating cells and their dissemination to distant organs and facilitate establishment of metastatic lesions. Hence, the central hypothesis of this proposal is that GRP/GRP-R signaling via AKT2 regulates resistance to conventional therapies and formation of metastatic foci, thereby, implicating a critical role for this axis in NB refractoriness and disease relapse. o examine this hypothesis, we propose the following three Specific Aims: 1) Determine the role of GRP-R/AKT2 signaling in inducing resistance to conventional therapies and tumor progression. 2) Determine the role of AKT2 in mediating the oncogenic effects of GRP/GRP-R in refractory and/or metastatic NB. 3) Determine the preclinical efficacy of targeting GRP-R/AKT2 in refractory and/or metastatic NB. Successful completion of our Aims will yield significant new knowledge regarding the mechanisms of GRP/GRP-R mediated NB refractoriness and disease relapse, including the role of cancer stem cell in metastasis as well as the extravasation of tumor cells and their interactions with TME. The aims proposed have direct translational relevance as they address an important molecular mechanism of resistance to conventional therapies and the TME. Finally, preclinical study will further provide important information on the safety and efficacy of novel therapeutics against virulent `high-risk' NB.